Purpose: :
The RGC-5 cell line is being increasingly used for studies intoganglion cell response to pathological insults. Although originallydescribed to be fully differentiated to a ganglion cell phenotypeafter treatment with succinyl concanavalin A (sConA), it hasbecome apparent that these cells are now less responsive tothis agent. Recently, the alternative compounds staurosporine(STSN) and trichostatin A (TCA) have also been described toterminally differentiate RGC-5 cells. We therefore comparedthe response of such cells to each of the three agents, in orderto definitively ascertain which treatment rendered a cellularphenotype closest to true ganglion cells.

Methods: :
RGC-5 cells were passaged when approximately 70% confluent intoappropriate vessels. After remaining in serum-free medium for24 hours, cells were treated for 3 days with either STSN (300nM),TCA (500ng/ml), sConA (50microg/ml) or vehicle (controls). Cellswere subsequently assayed for neuronal and ganglion cell markersby immunocytochemistry, immunoblotting and RT-PCR. In all cases,expression was compared with adult rat-derived ganglion cellsmaintained in culture for 14 days.

Results: :
Control, vehicle-treated RGC-5 cells expressed trace levelsof mRNA and proteins for the neuronal markers, tau, beta-tubulin,Neu-N, MAP2 and PGP9.5, as did the adult rat-derived ganglioncells. Although treatment with sConA had no effect on the expressionof these markers, STSN and TCA both markedly increased the levelsof beta-tubulin, PGP9.5 and MAP2, with the latter agent alsoincreasing the levels of tau. The effect of TCA was much moremarked than STSN in each case. No RGC-5 cells expressed theganglion cell markers, Brn-3, Thy-1 or neurofilament (either68kD or 200kD forms), or other retinal neurone markers suchas PKC-alpha, calretinin or calbindin. Interestingly, all cells,whether treated or not, expressed high levels of the neuronalprecursor protein, nestin. Expression levels of protein speciesinvestigated matched the levels of their appropriate mRNAs inall cases.

Conclusions: :
RGC-5 cells expressed low-levels of a number of neuronal butnot ganglion cell specific proteins and mRNAs. These were unaffectedby treatment with sConA, but STSN, and more particularly, TCAcaused specific upregulation of such markers. In conclusion,although none of the tested agents led to expression of ganglioncell-specific marker proteins or mRNAs, treatment with TCA representedthe best means by which to terminally differentiate RGC-5 cellsinto a true neuronal phenotype.